Single Protein Production in Yeast Cells

描述(由申请人提供)：以快速和廉价的方式解决蛋白质的三维结构的能力对于结构基因组学任务是重要的。蛋白质结构为基础科学和临床知识库增加了有价值的信息。在临床方面，这些结构可用于阐明重要的疾病相关蛋白的功能，用于识别影响人类健康的关键信号蛋白的抑制物或增强剂，或用于识别药物靶点。目前，细菌表达系统是最常用的利用核磁共振(NMR)和X射线结晶学进行结构研究的方法。我们最近在大肠杆菌中发现了一种名为MazF的ACA序列特异性内切核酸酶。我们的初步研究表明，具有MazF活性的细胞促进了来自不含ACA序列的mRNAs的高水平蛋白的表达。相反，由于宿主细胞mRNAs中含有丰富的ACA序列，因此只有微量的背景细胞蛋白质合成发生。该RO1建议的目的是基于这样一个假设，即将MazF内切核酸酶的独特性质应用于酵母细胞表达系统将提高一系列正确折叠的真核蛋白的有效表达和回收。MazF表达的独特性质将被用于开发酵母单蛋白生产(SPP)系统。这些属性应该有助于核磁共振和X射线结构研究，而不必实施蛋白质纯化步骤，并可能允许使用核磁共振直接显示活细胞中的蛋白质结构。目的1提出在酿酒酵母中建立基于MazF的SPP系统的实验。目的2将在酿酒酵母中开发的技术应用到克鲁维酵母的乳酸中，以努力扩大产品产量。在目标3中，将研究MazF在酵母细胞上表达的生理后果，以便利用SPP系统优化表达。最后，在目标4中，SPP系统将应用于真核蛋白质的子集，这些蛋白质在细菌表达时展开。在异核单量子相干分析中显示正确折叠的蛋白质随后将接受核磁共振结构确定。

DESCRIPTION (provided by applicant): The ability to solve the three dimensional structure of a protein in a rapid and inexpensive manner is instrumental to the Structural Genomics mission. Protein structures add valuable information to the basic science and clinical knowledge base. On the clinical side, these structures can be instrumental for illuminating the function of important disease related proteins, for identifying inhibitors or enhancers of key signaling proteins that influence human health or for identification of drug targets. Currently, bacterial expression systems are most commonly enlisted for structure studies using nuclear magnetic resonance (NMR) and X-ray crystallography. We have recently discovered an ACA sequence-specific endoribonuclease in E. coli called MazF. Our preliminary studies demonstrate that cells possessing MazF activity facilitate the expression of high levels of protein derived from mRNAs engineered without ACA sequences. In contrast, because of the abundance of ACA sequences in host cell mRNAs, only trace amounts of background cellular protein synthesis occurs. The aims of this RO1 proposal are based on the hypothesis that application of the distinctive properties of MazF endoribonuclease to yeast cell expression systems will enhance the efficient expression and recovery of an array of correctly folded eukaryotic proteins. The unique properties of MazF expression will be exploited for development of a yeast single protein production (SPP) system. These attributes should facilitate NMR and X-ray structural studies without having to implement a protein purification step and will likely allow for direct visualization of protein structures in living cells using NMR. Aim 1 proposes experiments for the development of a MazF-based SPP system in yeast Saccharomyces cerevisiae. Aim 2 applies the technology developed in yeast S. cerevisiae to Kluyveromyce's lactis in an effort to amplify product yields. In Aim 3, the physiological consequences of MazF expression on yeast cells will be studied in order to optimize expression using the SPP system. Finally, in Aim 4 the SPP system will be applied to a subset of eukaryotic proteins that are unfolded when expressed bacteria. Proteins that display proper folding upon Heteronuclear Single Quantum Coherence analysis will then be subjected to NMR for structural determination.